Details of experiments between P63104 and O60825
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: PFKFB2
Protein B fragment: 470-479
Protein B site: 14-3-3_pThr475
Protein B modification: (TPO)475
Protein B sequence: LSSSNTIRRP
Protein B construct: LSSSN(pThr)IRRP (crude synthetic peptide)
Average holdup BI: 0.59
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.33
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 4.03
Experiment series 2
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: PFKFB2
Protein B fragment: 461-470
Protein B site: 14-3-3_pSer466
Protein B modification: (SEP)466
Protein B sequence: RMRRNSFTPL
Protein B construct: RMRRN(pSer)FTPL (crude synthetic peptide)
Average holdup BI: 0.45
Holdup BI standard deviation: 0.21
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.2
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 3.79
Experiment series 3
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: PFKFB2
Protein B fragment: 478-487
Protein B site: 14-3-3_pSer483
Protein B modification: (SEP)483
Protein B sequence: RPRNYSVGSR
Protein B construct: RPRNY(pSer)VGSR (crude synthetic peptide)
Average holdup BI: -0.32
Holdup BI standard deviation: 1.54
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.16
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
The affinity was below the detection threshold of the assay.